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Genomics III: Methods, Techniques and Applications
Title
Genomics III: Methods, Techniques and Applications
Editor
iConcept Press
Price
USD$139.00
ISBN
978-1-922227-416
Clicks
17340

Chapter 15

Genomics III: Methods, Techniques and Applications

Strategies for Genetic Screening of Multiple Samples Using PCR-Based Targeted Sequence Enrichment

by Paola Benaglio and Carlo Rivolta

Viewed: 1834

Abstract

Next generation sequencing (NGS) has optimal applications in projects involving the analysis of large portions of DNA, such as the human genome or exome. Conversely, procedures allowing simultaneous sequencing of multiple samples for smaller DNA portions are not yet completely standardized and are still rather expensive. In this paper, we present two methods for concurrent sequencing of numerous samples. As a practical example, we take a mainstream application in medical genetics: the screening of single candidate genes in a cohort of patients affected by a hereditary disease. Both methods are applied to analyze DNA from ~100 patients for a 50 to 70 kb region, consisting in exons and introns of a target gene. Both methods take also advantage of long-range polymerase chain reaction (LR-PCR) as enrichment technique. In the first strategy, all the PCRs are pooled in a unique preparation and the detection of DNA variants is performed according to the estimated frequency of a single allele the pool. In the second strategy, each sample is tagged by a nucleotide barcode before the pooling. In this case, variation analysis is performed separately for each patient. We describe the workflow and the results of such procedures and discuss the defects and the advantages of each strategy, with the aim of providing useful guidelines for NGS-based molecular genetic investigations.

Author Details

Paola Benaglio
Department of Medical Genetics, University of Lausanne, Switzerland
Carlo Rivolta
Department of Medical Genetics, University of Lausanne, Switzerland

Citation

Paola Benaglio and Carlo Rivolta. Strategies for Genetic Screening of Multiple Samples Using PCR-Based Targeted Sequence Enrichment. In Genomics III: Methods, Techniques and Applications. ISBN:978-1-922227-416. iConcept Press. 0000.

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